The present invention relates to obtaining a natural mixture of conjugated equine estrogens which is depleted in phenolic urine contents and in non-conjugated lipophilic compounds from the group comprising non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, in particular androstane and pregnane steroids, and comparable non-conjugated compounds.
Estrogens are used in medicine for hormone replacement therapy. In particular, estrogen mixtures are used for the treatment and prophylaxis of the disorders of the climacteric period which occur in women after natural or artificial menopause. In this case, natural mixtures of conjugated estrogens such as are found in the urine of pregnant mares, hereafter referred to as natural mixtures of conjugated equine estrogens, have proved particularly effective and readily compatible.
The dissolved solids content in the urine of pregnant mares (=pregnant mares' urine, abbreviated hereafter as “PMU”) can naturally fluctuate within wide ranges, and may generally lie in a range of 40 to 90 g dry matter per liter. In addition to urea and other usual urine contents, phenolic constituents are contained in the solids content of the PMU in quantities of about 2 to 5% by weight relative to the dry matter. These phenolic constituents include cresols and dihydro-3,4-bis[(3-hydroxyphenyl)methyl]-2(3H)-furanone, known as HPMF. These may be present in free or conjugated form. The PMU contains a natural mixture of estrogens which is largely present in conjugated form, e.g. as sulfuric acid semi-ester sodium salt (abbreviated hereafter as “sulfate salt”). The content of conjugated estrogens (calculated as estrogen sulfate salt) may be between 0.1 and 0.5% by weight, relative to the dry matter. In addition, further lipophilic compounds may be present in the solids content of the PMU, the quantities of which compounds can fluctuate within wide ranges and cannot be predicted. These lipophilic compounds originate predominantly from the plants ingested as food by the pregnant mares and comprise above all various flavonoid, isoflavonoid and norisoprenoid derivatives and comparable compounds, such as for example formononetin, genistein, daidzein, biochanin A, equol and coumestrol. These lipophilic compounds originally of plant origin may be present in the urine in conjugated or in free (non-conjugated) form. The lipophilic constituents furthermore occurring in the solids content of the PMU also include non-conjugated steroid derivatives; of these in particular the androstane and pregnane steroids, but also non-conjugated estrogen derivatives, should be mentioned.
Extracts containing natural mixtures of conjugated estrogens are usually obtained either by means of a solid-phase extraction method or by a method based on various liquid-liquid extraction steps with organic solvents which are not miscible, or only slightly miscible, with water. Generally speaking, in order to be able to be used as active substance constituent for pharmaceuticals, the natural mixture of conjugated estrogens which is obtained must meet certain pharmaceutical specifications, for example meet the specification laid down in the USP (United States Pharmacopeia) or European Pharmacopoeia. For example, certain limit values must be observed with regard to the content of conjugated estrogens relative to the dry matter.
U.S. Pat. Nos. 2,551,205 and 2,429,398 describe a method for preparing a water-soluble estrogen preparation from PMU, in which first an aqueous concentrate is obtained by adsorption on activated carbon or other suitable adsorber materials, elution with a water-miscible organic solvent, such as pyridine, and subsequent removal of the solvent, which concentrate contains the major part of the water-soluble estrogen constituents of the PMU originally used. Whereas in U.S. Pat. No. 2,429,398 the concentrate is further purified by extraction with benzene and/or ether, U.S. Pat. No. 2,551,205 discloses acidulating the concentrate to a pH value of between 2 and 6, preferably between 4 and 5, and then rapidly extracting it with a organic solvent which is only slightly miscible with water selected from the group consisting of aliphatic, aromatic or alicyclic hydrocarbons (e.g. hexane, benzene, toluene, cyclohexane) or chlorinated hydrocarbons (e.g. chloroform, ethylene dichloride, trichloroethylene, carbon tetrachloride, chlorobenzene), in order to separate undesirable substances such as fats, oils, free phenolic constituents and the non-conjugated steroids by transfer into the organic phase. Finally, the aqueous phase is stabilized by neutralization. U.S. Pat. No. 2,551,205 recommends further purifying the resulting extract by subsequent extraction steps and precipitation operations. Overall, after carrying out the method described in U.S. Pat. No. 2,551,205, a yield of only about 80% of the estrogen constituents of the concentrate used is obtained.
U.S. Pat. No. 2,565,115 describes extraction of the conjugated estrogens from PMU with acetone. No statement is made about the purity of the resulting estrogen fraction.
U.S. Pat. No. 2,696,265 describes a method in which initially the estrogens are extracted with an aliphatic alcohol or ketone, such as hexanol, cyclohexanol or cyclohexanone. The estrogens pass into the organic phase and are then further purified; inter alia, an aqueous phase containing the estrogens is adjusted to a pH value of 4 with hydrochloric acid and extracted with ethylene dichloride.
U.S. Pat. No. 2,834,712 discloses a method for preparing estrogen mixtures of significant purity and low toxicity which is based on a large number of individual extraction steps with different solvents and the setting of different pH values. In that method, large volumes of solvents such as hexane and benzene are used. Thus for example in one step an already-purified concentrate is dissolved in water, adjusted to a pH value of approximately 5.0 with hydrochloric acid, and extracted with benzene and then with ether, in order to separate out the phenolic constituents.
International patent application no. WO 01/27134 describes a comparatively simple method of extracting conjugated estrogens from PMU. After the addition of a salt, such as sodium chloride, the PMU is extracted with at least the same volume percent of an organic solvent, such as ethyl acetate, whereupon the conjugated estrogens pass into the organic phase. The organic phase is separated and dried in order to obtain the extract. No statements are made in WO 01/27134 about the purity of the extract of conjugated estrogens which is obtained.
With the liquid-liquid-extraction method described above and known from the prior art, however, a number of problems occur, such as vigorous foaming, sediment formation, emulsification and poor phase separation. Generally several extraction steps are required, which results in losses and only partial recovery of the estrogen content. Furthermore, these extraction methods require large volumes of solvents, some of which may be hazardous. Furthermore, in the patent specifications listed above no statements are made either about the content of non-conjugated lipophilic constituents, such as for example non-conjugated flavonoid, isoflavonoid and norisoprenoid derivatives and comparable non-conjugated compounds, or also non-conjugated steroids, in particular androstane and pregnane steroids, in the resulting products, or about separation of these constituents. These methods known from the prior art either provide no satisfactory results with regard to the yield or with regard to the purity of the extract obtained, measured by the total hormone content obtained relative to the dry matter, or they are based on a large number of different method steps and the use of large volumes of organic solvents, some of which may be hazardous.
Furthermore various solid-phase extraction methods are known from the prior art for obtaining a natural mixture of conjugated equine estrogens largely depleted in phenolic urine contents.
Thus, international patent application no. WO 98/08526 describes a method with which a largely cresol-free and HPMF-free mixture, which is depleted in phenolic urine contents and contains the natural estrogen content of the PMU practically completely can be obtained in a solid-phase extraction on a semipolar, in particular non-ionic semipolar, polymeric adsorption resin. International patent application no. WO 98/08526 describes a similar method in which silica gel is used as adsorber material in the solid-phase extraction. Also, Chinese patent application CN 1308083 describes a comparable method in which polar adsorption resins containing cyano groups are used. Furthermore, published U.S. patent application Ser. No. US 2002/0156303 describes a method in which the PMU before purification over a polystyrene adsorption resin is first treated with an alkaline solvent and is pre-purified by filtration. The resulting extracts are suitable as starting materials for preparing pharmaceuticals which contain the natural mixture of conjugated estrogens from PMU as active substance constituent.
The pharmaceutical specification requirements laid down, for example the limit values to be observed with regard to the content of conjugated estrogens relative to the dry matter, are normally met by the mixtures of conjugated estrogens obtained from PMU in accordance with the method of WO 98/08526 or the method of WO 98/08525. In particular, it is possible, using the method disclosed therein, to obtain a largely cresol-free and HPMF-free product which is depleted in phenolic urine contents. It has however turned out that in addition to the desired content of conjugated estrogens, the resulting dry matter also may contain non-conjugated lipophilic compounds. The non-conjugated lipophilic compounds include, for example, various non-conjugated flavonoid, isoflavonoid and norisoprenoid derivatives and comparable non-conjugated compounds, such as for example formononetin, genistein, daidzein, biochanin A, equol and coumestrol, but also non-conjugated steroids, in particular androstane and pregnane steroids, and non-conjugated estrogens. The foregoing list should not be regarded as definitive. The presence of these non-conjugated lipophilic compounds in the mixture of conjugated estrogens obtained from the PMU cannot be standardized, but both the content and the composition of the free and conjugated lipophilic compounds varies, for example, depending on the feed ingested by the pregnant mares.
Although the composition of the natural mixture of conjugated equine estrogens does not change due to the additional presence of the non-conjugated lipophilic compounds, the content of the conjugated equine estrogens relative to the dry matter can be reduced. A higher concentration of the active substances, i.e. the conjugated equine estrogens, in the product extract could be achieved by deliberate separation of the non-conjugated lipophilic constituents. Furthermore, separation of the non-conjugated lipophilic compounds ensures a more uniform composition of individual extract batches, since in this way the non-conjugated lipophilic constituents, the content and composition of which in the PMU can vary depending on the seasonally changing type of food ingested by the pregnant mares, are eliminated, and thus the resulting extracts all have a comparable content of conjugated equine estrogens relative to the dry matter. Furthermore, separation of the non-conjugated lipophilic compounds may be advantageous in order to obtain a uniform physiological activity spectrum. For example, it may be useful to separate out possibly present, non-conjugated lipophilic compounds, which may themselves have inherent physiological effects, from the natural mixture of conjugated equine estrogens.
One possible way of separating the undesirable, non-conjugated lipophilic compounds would be, for example, to subject the natural mixtures of conjugated equine estrogens obtained using the known solid-phase extraction methods set forth above to a separate liquid-liquid extraction with a suitable organic solvent which specifically extracts the undesirable, non-conjugated lipophilic compounds, without resulting in a loss of conjugated equine estrogens. Such a method is broadly described in the pending international patent application PCT/EP 03/50703.